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Abstract：
Avian malaria and related haemosporidians (Plasmodium, [Para]Haemoproteus and Leucocytoozoon) represent an exciting multihost, multiparasite system in ecology and evolution. Global research in this field accelerated after the publication in 2000 of PCR protocols to sequence a haemosporidian mitochondrial (mtDNA) barcode and the development in 2009 of an open-access database to document the geographic and host ranges of parasite mtDNA haplotypes. Isolating haemosporidian nuclear DNA from bird hosts, however, has been technically challenging, slowing the transition to genomic-scale sequencing techniques. We extend a recently developed sequence capture method to obtain hundreds of haemosporidian nuclear loci from wild bird samples, which typically have low levels of infection, or parasitemia. We tested 51 infected birds from Peru and New Mexico and evaluated locus recovery in light of variation in parasitemia, divergence from reference sequences and pooling strategies. Our method was successful for samples with parasitemia as low as ~0.02% (2 of 10,000 blood cells infected) and mtDNA divergence as high as 15.9% (one Leucocytozoonsample), and using the most cost-effective pooling strategy tested. Phylogenetic relationships estimated with >300 nuclear loci were well resolved, providing substantial improvement over the mtDNA barcode. We provide protocols for sample preparation and sequence capture including custom probe sequences and describe our bioinformatics pipeline using atram 2.0, phyluce and custom Perl/Python scripts. This approach can be applied to thousands of avian samples that have already been found to have haemosporidian infections of at least moderate intensity, greatly improving our understanding of parasite speciation, biogeography and evolutionary dynamics.
摘要：
禽疟疾和相关血吸虫(疟原虫，[第]血液变形杆菌和白细胞）代表令人兴奋的多宿主，生态学和进化中的多寄生虫系统。在2000年发布PCR协议以测序血球线粒体（mtDNA）条形码以及2009年开发开放访问数据库以记录寄生虫mtDNA单倍型的地理和宿主范围之后，该领域的全球研究加速。从鸟类宿主中分离血孢子虫核DNA，然而,在技术上具有挑战性，减缓向基因组规模测序技术的过渡。我们扩展了最近开发的序列捕获方法，以从野生鸟类样本中获得数百个血孢子虫核基因座，通常具有低水平的感染，或者寄生虫血症.我们测试了来自秘鲁和新墨西哥州的51只感染鸟类，并根据寄生虫血症的变化评估了基因座恢复情况，与参考序列和池化策略的差异。我们的方法成功用于寄生虫血症低至〜0.02％（感染的10,000个血细胞中有2个）和mtDNA差异高达15.9％（一个白细胞样本）的样品，并使用经过测试的最具成本效益的池策略。>300个核基因座估计的系统发育关系得到了很好的解决，提供对mtDNA条形码的实质性改进。我们提供了用于样品制备和序列捕获的协议，包括自定义探针序列，并使用atram2.0，phyluce和自定义Perl/Python脚本描述了我们的生物信息学管道。这种方法可以应用于已经发现至少中等强度的血孢子虫感染的数千个禽类样本，大大提高了我们对寄生虫物种的理解，生物地理学和进化动力学。
